Project Summary Planarian flatworms are famous for their ability to rapidly regenerate new heads or even entire organisms from a tiny fragment of the animal. Planarian regeneration involves a population of proliferative cells (neoblasts) that include pluripotent adult stem cells (cNeoblasts) and that can produce every cell of the adult animal. Despite centuries of fascination with regeneration, mechanistic explanations await elucidation. The broad, long-term objectives of this proposal are to use planarians as a model system to identify and understand the molecular mechanisms that regulate stem cells to promote regeneration. The specific aims are: 1) To determine the mechanism of tissue-specific regeneration, 2) To determine how progenitors organize into a regenerated structure, and 3) To identify in vivo regulatory mechanisms of stem cell fate specification. Stem cells and regenerative biology are the subjects of recent and intense interest for regenerative medicince. In addition, the misregulation of stem cells may be central to many types of cancer. A newly developed arsenal of tools for molecular genetic study of stem cell biology in planarians now exists. For example, systematic gene perturbation with RNA interference (RNAi) is possible and the planarian genome has been sequenced. Greater than half of planarian genes have counterparts in the human genome; therefore, planarian studies should identify conserved stem cell regulatory mechanisms. Aim #1 will determine the mechanisms by which a stem cell response can bring about the specific regeneration of exactly those tissues that were missing. This is a fundamental problem of wound repair and regeneration in most organisms, including humans. Aim #2 will determine the mechanisms that lead regenerative progenitors to incorporate into the correct tissues at the correct locations. We will utilize the study of planarian eye progenitors for this work. These eye progenitors home to the exact location where eyes should form, and are incorporated into existing eyes. Aim #3 will study the mechanisms of stem cell fate specification. Some processes must exist to regulate stem cell number in planarians, and their choice to make more stem cells or to make differentiated cells. We will study how contextual cues regulate this process by investigating candidate neoblast regulatory cells and by study of the specialization of planarian stem cells into regenerative progenitors of the nervous system. Successful completion of proposed aims will greatly advance our understanding of the mechanistic basis for regeneration and advance planarians as a model system for the study of genes conserved in humans in stem cell and regenerative biology, areas of great importance in human health.